Human CHD4 (chromodomain helicase DNA-binding protein 4) plays a critical role in the regulation of chromatin structure and the control of B- and T-cell differentiation. CHD4 is an ATPase and a major subunit of the nucleosome remodeling and deacetylase (NuRD) complex, essential in the transcriptional repression, however its role in the complex remains elusive. CHD4 contains tandem plant homeodomain (PHD) fingers of unknown function. Recent studies from our laboratory demonstrate that both PHD fingers recognize histone H3, revealing a novel link between NuRD-mediated signaling and chromatin remodeling. We hypothesize that the tandem PHD1/2 fingers of CHD4 bind cooperatively to N-terminal tails of histone H3, recruiting and/or stabilizing the NuRD complex at chromatin. We seek to elucidate the molecular mechanism, the structural basis and the functional significance of the interaction of CHD4 PHD1/2 with H3. By establishing the biological roles of the PHD fingers and obtaining the atomic-resolution structures of their complexes, we will learn how the CHD4/NuRD machinery associates with chromatin. The specific aims of this project are: (1) to elucidate the functional and structural relationship between PHD1 and PHD2 of CHD4 and histone tails, and (2) to establish the molecular mechanism of the chromatin targeting by the tandem PHD1/2 fingers. The atomic-resolution structures of the individual and tandem CHD4 PHD fingers in complex with histone tail peptides will be determined by NMR spectroscopy or X-ray crystallography. The histone specificities and binding affinities will be examined by western blot assays, peptide library screening, NMR, and fluorescence spectroscopy. The binding site residues will be mutated and the mutant proteins will be tested in vitro by NMR and pull-down experiments and in vivo in a B lymphocyte cell line following depletion of the endogenous protein using shRNA. We will utilize chromatin immunoprecipitation, chromatin accessibility and transcriptional activation assays in this context to assess how the PHD fingers contribute to functions of NuRD. This research will shed light on the role of the tandem PHD fingers in bridging the CHD4/NuRD complex with chromatin and will lead to a better understanding of the general mechanisms of transcription regulation and chromatin remodeling. These experiments will also provide important insights into CHD4/NuRD-dependent signaling pathways that may constitute new targets for therapeutic manipulation.